Mutations in the tetramer interface of human glucose-6-phosphate dehydrogenase reveals kinetic differences between oligomeric states

Article abstract of DOI:10.1002/1873-3468.12638

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the oxidation of glucose-6-phoshate to 6-phospho-gluconolactone with the concomitant reduction of NADP⁺ to NADPH. In solution, the recombinant human G6PDH is known to be active as dimers and tetramers. To distinguish between the kinetic properties of dimers and tetramers of the G6PDH is not trivial. Steady-state kinetic experiments are often performed at low enzyme concentrations, which favor the dimeric state. The present work describes two novel human G6PDH mutants, one that creates four disulfide bonds among apposing dimers, resulting in a ‘cross-linked’ tetramer, and another that prevents the dimer to dimer association. The functional and structural characterizations of such mutants indicate the tetramer as the most active form of human G6PDH.

Full text of DOI:10.1002/1873-3468.12638

Article type : Research Letter
Mutations in the tetramer interface of human glucose-6-phosphate
dehydrogenase reveals kinetic differences between oligomeric states
Americo Tavares Ranzani1,2 and Artur Torres Cordeiro2,*.
1. Institute of Biology, University of Campinas, Campinas-SP, Brazil.
2. Brazilian Biosciences National Laboratory, Brazilian Center for Research in Energy and
Materials, Campinas-SP, Brazil.
* corresponding author address:
Center for Research in Energy and Materials
Brazilian Bioscience National Laboratory
R. Giuseppe Maximo Scolfaro, 10000.
Campinas – SP
ZIPCODE: 13083-100
BRAZIL
Phone: +55 19 3512-1121
Fax: +55 19 3512-1106
Email: artur.cordeiro@lnbio.cnpem.br
KEYWORDS: glucose-6-phosphate dehydrogenase / disulfide bond / oligomeric state
ABSTRACT
Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the oxidation of glucose-6-phoshate
to 6-phospho-gluconolactone with the concomitant reduction of NADP+ to NADPH. In solution,
the recombinant human G6PDH is known to be active as dimers and tetramers. To distinguish
between the kinetic properties of dimers and tetramers of the G6PDH is not trivial. Steady-
state kinetic experiments are often performed at low enzyme concentrations, which favor the
dimeric state. The present work describes two novel human G6PDH mutants, one that creates
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four disulfide bonds among apposing dimers, resulting in a “cross-linked” tetramer, and
another that prevents the dimer to dimer association. The functional and structural
characterizations of such mutants indicate the tetramer as the most active form of human
G6PDH.
INTRODUCTION
The enzyme Glucose-6-phosphate dehydrogenase, E.C. 1.1.1.49 (G6PDH) catalyzes the
first step of the pentose phosphate pathway (PPP), which consists in the oxidation of glucose-
6-phoshate (G6P) to 6-phospho-gluconolactone with the concomitant reduction of NADP+ to
NADPH. G6PDH plays a central role in cellular homeostasis by supplying NADPH for several
biosynthetic processes and to maintenance of redox balance. G6PDH deficiency is the most
common human enzymatic defect disorder; it causes hemolytic anemia triggered by exposure
of red blood cells to oxidant agents (1). Despite this negative effect, G6PDH deficiency confers
some level of resistance against malaria, being found in higher frequency in regions where
malaria is endemic (2). Although G6PDH is considered a “housekeeping” enzyme, its
expression level, intracellular translocation and post-translation (phosphorylation)
modification have been demonstrated to be regulated, both positively and negatively, by
different signaling factors (revised by Stanton, 2010 (3)). Altered G6PDH regulation can affect
cellular metabolism and propagation, with direct implication to cancer (4), metabolic
syndrome and vascular diseases progression (5).
The active forms of the human G6PDH are associated to dimeric and tetrameric states,
but not to monomers. Equilibria exist between tetramers to dimers and dimers to monomers,
but not between tetramers directly to monomers. Moreover, NADPH shifts the equilibrium
from active dimers to inactive monomers (6). The equilibrium between dimer and tetramer is
affected by ionic strength, pH and enzyme concentration; pH below 7.2, low ionic strength,
high protein concentration favor the tetrameric state (7). Additionally, the binding of a
structural NADP+ to a site different from the catalytic one, also favors the tetrameric
conformation (8). Crystallographic structures of recombinant human G6PDHs present the
protein at the tetrametric state, even when crystals were grown at pH 5.8 (9).
Usually standard G6PDH steady-state kinetic experiments are performed at low
enzyme concentration by measuring the fluorescence or absorbance relative to NADPH
production. Once G6PDH dimer is predominant at low protein concentration, one can assume
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that data retrieved from such kinetic experiments are related to the dimeric form of the
enzyme. In order to compare kinetic parameters between dimers and tetramers it would be
necessary to establish a “cross-linked” G6PDH tetramer that would resist dissociation once
diluted during the kinetic experiments.
In the present work we describe the design of two new G6PDH mutants, one that
prevents the tetramer formation and another that stabilizes the tetramer by the introduction
of four disulfide bridges between adjacent dimers. The kinetic characterization of those
mutants indicates relevant differences between dimers and tetrames of human G6PDH.
MATERIALS AND METHODS,
Site-directed mutagenesis and preparation of recombinant G6PDHs
The cloning of the expression vector pET_SUMO_HsΔG6PDH encoding for a short
construction of HsG6PDH (HsΔG6PDH, residues 29 to 511) with a N-terminal His-SUMO tag was
described elsewhere (10). The single HsΔG6PDH mutants A277C and E347A
(HsΔG6PDH_A277C and HsΔG6PDH_E347A, respectively) were generated using the
QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene), following the manufacturer’s
instructions and with the oligonucleotides: 5’-GGCCATGGAGAAGCCCTGCTCCACCAACTCAG-3’
and
5’-CTGAGTTGGTGGAGCAGGGCTTCTCCATGGCC-3’
for
A277C
and
5’-
CTATGTGGAGAATGCGAGGTGGGATGGGG-3’ and 5’- CCCCATCCCACCTCGCATTCTCCACATAG-3’
for E347A (nucleotides underlined indicate the mutation positions). Mutations were verified by
automated sequencing of both mutant constructions in a HITACH 3130X Genetic Analyzer
(Applied Biosystems).
The expression and purification of both HsΔG6PDH mutants followed the protocol
previously stablished for HsΔG6PDH (10). Briefly, E. coli BL21 (DE3) cells harbouring either
HsΔG6PDH_A277C or HsΔG6PDH_E347A vectors were grown in ZYM5052 autoinduction
medium (11) for 48h at room temperature and 250 rpm. Cells were harvested, lysed by
sonication, clarified by centrifugation and submitted to metal affinity chromatography (Ni-
NTA, Invitrogen). The His-SUMO tag was cleaved by addition of ULP-1 protease nickel charge
resin and HsΔG6PDH mutants were recovered at resin wash step. Both HsΔG6PDH mutants
were then concentrated and submitted to a final size exclusion chromatography step.
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Size exclusion chromatography
The preparative and analytical size exclusion chromatography were performed using
the columns Superdex 200 16/60 (GE Healthcare) and Superdex 200 10/30 (GE Healthcare),
respectively, in an AKTA purifier FPLC system (GE Healthcare). The proteins were applied at 4
mg/mL (71 µM) on preparative column. The fractions of the main peak were collected,
concentrated and subjected to analytical chromatography. For HsΔG6PDH, two different
dilutions were applied: 4 mg/mL (71 µM) and 0.4 mg/mL (7.1 µM). While for
HsΔG6PDH_E347A and HsΔG6PDH_A277C, each one was applied at 4 mg/mL (71 µM) and 0.4
mg/mL (7.1 µM), respectively. The proteins were eluted in 50 mM Tris-HCl pH 8.0 and 150 mM
NaCl. The analytical column was calibrated using a molecular weight (MW) protein markers kit
(MWGF200, Sigma-Aldrich): β-amilase (200 kDa), Alcohol Dehydrogenase (150 kDa), Albumin
(66 kDa), Carbonic anhydrase (29 kDa) and Cytochrome C (12.4 kDa). The void volume (Vo) was
determined by Blue Dextran. The MW markers chromatography was performed at the same
conditions of HsΔG6PDH. The calibration curve (Fig. S1) was done following manufacturer’s
recommendations.
Protein crystallization and structure determination
The first crystals of HsΔG6PDH_A277C were obtained at 291 °C by the hanging-drop
vapor diffusion method, following the crystallization conditions published elsewhere (12). The
crystal used to solve the structure was grown from drops made of well buffer containing 100
mM Tris-HCl pH 9.0, 200 mM MgCl2, 13% PEG 4000 and 20% glycerol and a protein solution
comprised of 20 mg/mL HsΔG6PDH_A277C, 2mM NADP+, 10mM G6P, 50 mM Tris-HCl pH 8.0
and 150 mM NaCl. The crystal was flash-frozen and collected at 100 K, at X06DA beamline
from the Swiss Lightsource (Villigen, Switzerland), using a PILATUS 2M-F detector (DECTRIS).
Diffraction data were indexed and integrated using XDS (13), and scaled by SCALA (14). The
structure was solved by molecular replacement, using the HsΔG6PDH structure (PDB: 2BH9) as
search model, using MOLREP (15). Restrained refinement was performed using REFMAC (16)
and manual rebuilding in Coot (17). The diffraction data and refinement statistics are shown at
table 1.
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Enzyme kinetics
G6PDH activity was monitored by measurement of NADPH absorbance at 340 nm,
recorded in an Envision microplate reader (Perkin-Elmer). The assays were performed at 25 °C,
in 96 well microplates, final reaction volume of 0.2 ml, in reaction buffer (50 mM Tris-HCl pH
8.0, 150 mM NaCl). The apparent-Km for G6P was determined measuring NADPH formation
rates at different concentrations of G6P (1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.8125
app
µM) and a fixed NADP+ concentration (200 µM). The Km for NADP+ was determined at
different concentrations of NADP+ (200, 100, 50, 25, 12.5, 6.25, 3.125 µM) and a fixed G6P
concentration (1 mM). A calibration curve of NADPH absorbance value in different
concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125 µM) was used to calculate the NADPH
formation velocity from the absorbance measurements. The resultant curves were fitted to
app
Michaelis-Menten equation to determine the Km
and maximum velocity. For DHEA
inhibition assay, the enzyme activity was measured in varying concentrations of inhibitor (80,
40, 20, 10, 5, 2.5 and 1.25 µM, 2 % DMSO) with saturating substrate condition (1 mM G6P and
200 µM NADP+). The IC50 values were calculated by fitting the normalized activity measured (%
Activity) at different inhibitors concentration to the sigmoidal curve (Eq. 1). For enzyme activity
normalization, reaction velocity in the absence of inhibitor or substrate were used as 100 and
0 %, respectively. In all assays, the enzyme concentration used was 10, 2.5 and 20 nM for
HsΔG6PDH, HsΔG6PDH_A277C and HsΔG6PDH_E347A, respectively. The data analysis was
done using GraphPad Prism 5 (GraphPad Software).
100
% Activity =
Eq. 1
ꢊ
ꢀ
ꢁꢂꢃꢄꢅꢆꢇ
ꢈꢉ
1 + 10
X: logarithm of inhibitor concentration.
Thermal Stability
The thermal stability of HsΔG6PDHs was assessed by differential scanning fluorimetry
using SYPRO Orange (Sigma-Aldrich) as fluorescent dye (18). 25 µL of 5 µM of protein with 5X
of SYPRO Orange (in 50 mM Tris-HCl pH 8.0 and 150 mM NaCl) was dispensed in a 96-well
microplate. The data were taken on a 7500 PCR Real-Time System (Applied Biosystems),
measuring the SYPRO Orange fluorescence (filters recommended by manufacturer) in a
temperature range of 20 °C to 96 °C, in steps of 1 °C/ minute. The melting temperature (TM)
was retrieved by fitting the normalized fluorescence (% Fluorescence) to sigmoidal curve (Eq.
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2), using GraphPad Prism 5. For data normalization, it was considered as 0 % the lowest
fluorescence intensity value before the denaturation, and 100 %, the highest value after
denaturation. All assays were performed in triplicate.
100
% Fluorescence =
Eq. 2
ꢀ
ꢊ
ꢃꢄꢅꢋ ꢂꢁ .ꢍ
ꢌ
1 + 10
X: logarithm of temperature; n: Hill slope.
RESULTS AND DISCUSSION
HsG6PDH mutants design
According to Au and co-authors (19), the tetramer interface of the wild type HsG6PDH
buries only 706 Å2 per monomer and involves 15 residues, out of which 11 are charged
residues. The tetramer is formed by the interaction of dimer AB to CD, and four salt bridges
(K275(chain-A)/E347(chain-D), K275(chain-B)/E347(chain-C), E287(chain-A)/K290(chain-D) and
E287(chain-B)/K290(chain-C)) contribute to stabilize the tetrameric state (Fig. 1). The
electrostatic nature of the tetramer interface explains its sensitive to pH, ionic strength and
protein concentration. In order to disrupt the tetramer the residue glutamic acid 347 was
mutated to an alanine (E347A). Inspection of residues in the tetramer interface leads us to
hypothesize that mutation of alanine 277 to a cysteine would bring its sulfur atom in a
favorable orientation to form a disulfide bond with cysteine 294.
Oligomeric state of HsΔG6PD mutants
Preparative size exclusion chromatography of HsΔG6PD at 4 mg/mL returns two peaks
with elution volumes equivalent to tetrameric and dimeric states (Fig. 2A). When samples
collected from the peak corresponding to the tetrametic state are pooled together, re-
concentrated to 4 mg/mL and loaded into an analytical column, we observed an asymmetrical
peak with elution volume corresponding to a trimeric state (Fig.2B solid line). This effect may
be related to the rapid tetramer-dimer equilibrium, generating an intermediate apparent MW.
The same effect was already observed for HsG6PDH at 1 mg/mL by Wang et al. (8). However, if
the same sample is loaded at lower concentration (0.4 mg/mL), a single peak appears with an
elution volume characteristic of the dimeric state (Fig. 2B, dashed line). When the same
procedure is applied to the HsΔG6PD_ E347A mutant no signal of tetramer is observed in the
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preparative column, but only the dimer (Fig. 2C). Moreover, when samples collected from the
dimeric peak are concentrated to 4 mg/ml and loaded in the analytical column the mutant
remains as in the dimeric state (Fig. 2D). For the mutant HsΔG6PD_A277C, an increase in the
tetramer fraction at the preparative size exclusion chromatography is observed when
compared to HsΔG6PD (Fig. 2E) and the mutant remains as a tetramer even after a 10-fold
dilution (Fig. 2F). A minor dimeric fraction of HsΔG6PD_A277C is always observed in the
preparative run, most probably due to troubles in disulfide bridge formation in bacterial
cellular environment.
Structure of HsΔG6PD_A277C
Crystals for the mutant HsΔG6PD_A277C were obtained in the same crystallization
condition described for HsΔG6PD (12). The protein crystallized in space group F222, with one
monomer in the asymmetric unit. The tetramer is formed by the crystallographic 222
symmetry. One G6P is bound to the active site, at the same orientation of previous
characterized complex. The present structure revealed a right-handed disulfide bond between
cysteine 277 and 294, as clearly seen in the electron density (Fig. 3). The monomer structure is
highly conserved with the published HsΔG6PD:G6P structure (PDB: 2BHL), with an average
RMSD of 0.39 Å and 0.71 Å for the main chain and side chain, respectively.
Catalytic properties of HsΔG6PD mutant
The measurement of steady state kinetic parameters require the enzyme to be in a
concentration much lower than its substrates, usually in the nanomolar range. Under such
condition, human G6PDH will be predominantly in dimeric state, but a minority of tetramers
might remain. In order to access the catalytic proprieties of distinct oligomeric states from
HsΔG6PD, we stablished two new mutants that can be considered to exist exclusively as
dimers or tetramers at low protein concentration conditions. Comparison of kinetic
parameters among mutants and wildtype G6PDH shows a slight raise in Km values for the
HsΔG6PD_A277C (Table 2). While a pronounced increase is observed for kcat parameter. These
alterations lead to a gain in catalytic efficiency of HsΔG6PD_A277C, as observed by comparison
of its turnover rates to that of HsΔG6PD_E347A and wildtype.
An important feature of the human G6PDH is its uncompetitive inhibition by steroids
like dehydroepiandrosterone (DHEA). Although that was first described in 1960 (20), the
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precise location of the steroid binding site remains unknown. DHEA inhibition was assessed for
the mutants and no significant difference on IC50 values were observed (Table 2). That
suggests the steroid inhibition site was not affected by A277C neither E347A mutations and it
must exist independently of the ability of G6PDH to form tetramers.
Stability
The differential scanning fluorimetry assay shows a considerable gain of thermal
stability for the HsΔG6PD_A277C in comparison to HsΔG6PD_E347A and the wildtype (Table
3). The melting temperature of the HsΔG6PD_A277C is higher than ten degrees when
compared to the other proteins here evaluated. This is an additional feature of this G6PDH
mutant that may be explored towards the development of improved enzymatic reagents.
CONCLUSION
The functional and physiological significance of the interconversion of dimer-tetramer
of active HsG6PDH is elusive. The measurement of the tetramer enzymatic activity by steady-
state kinetics is hampered by the fact that the protein dilution in the assay favors the dimeric
state. Here we present HsG6PDH mutants that display just one oligomeric state, enabling the
study of each form independently. The point mutation E347A prevents the tetramer formation
even at high protein concentration, while A277C mutation results in the formation of an
interchain disulfide bridge with cysteine 294. This last single mutation leads to the
establishment of four disulfide bonds in the tetramer interface, making it stable at low protein
concentration. The comparison of the kinetic proprieties of the new mutants indicates that the
tetramer is at least fourfold more efficient than the dimer. Moreover, the incorporated
disulfide bonds make the tetrameric mutant less susceptible to thermal denaturation. Lastly,
the designed mutants are valuable tools for further studies to elucidate the role of the
oligomeric state in the function of HsG6PDH.
ACKNOWLEDGEMENTS
We thank Marcio V. B. Dias (University of Sao Paulo) for assistance in the data
collection and Sao Paulo Research Foundation for funding (grant: 2012/23682-7).
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FIGURES LEGENDS
Figure 1: Tetramer organization of HsG6PDH. The tetramer is formed by the dimers AB and CD.
In the boxes are shown the salt bridges which stabilize the tetramer. Also is highlighted the
proximity between alanine 277 and cysteine 294 in the tetramer interface. (The figure was
prepared using the structure of PDB entry 2BH9).
Figure 2: Size exclusion chromatography of HsΔG6PDH mutants. A, C and E: Preparative size
exclusion of HsΔG6PDH, HsΔG6PDH_E347A and HsΔG6PDH_A277C, respectively. In grey, the
fractions recovered and submitted to analytical size exclusion. B, D and F: Analytical size
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exclusion of HsΔG6PDH, HsΔG6PDH_E347A and HsΔG6PDH_A277C, respectively. The apparent
MW calculated for HsΔG6PDH_E347A, HsΔG6PDH_A277C and HsΔG6PDH at 4 mg/mL (71 µM)
and 0.4 mg/mL (7.1 µM) were 109, 231, 157 and 100 kDa, respectively. The used calibration
curve is at Supporting Information (Fig. S1). The theoretical MW of HsΔG6PDH monomer is
56.1 kDa. Dotted lines: HsΔG6PDH_A277C tetramer peak (11.58 mL) and HsΔG6PDH_E347A
dimer peak (13.29 mL).
Figure 3: Disulfide bridge in the tetramer interface of the mutant HsΔG6PDH_A277C. A: The
intermolecular disulfide bond formed by cysteines 277 and 294. B: Another orientation of the
right handed disulfide bridge. The backbone of monomer A and D are shown in green and grey,
respectively. In blue, 2Fo – Fc electron density map, contoured at 1 σ.
TABLES
Table 1: Diffraction data and refinement statistics of HsΔG6PDH_A277C.
Diffraction data statistics
Wavelength (Å)
Space group
0.97643
F222
Unit-cell parameters (Å)
Resolution (Å)
No. of reflections/unique reflections
Completeness (%)
Rpim (%)
a = 59.10 / b = 177.82 / c = 216.47
46.23 – 2.65
155303/16889
100.0
7.0 (58.4)
Mean I/σ
17.2 (1.7)
CC1/2
0.998 (0.579)
Refinement statistics
Rfactor/Rfree (%)
Rmsd bond lengths (Å)
Rmsd bond angles (°)
Number of atoms refined
Protein
18.9/23.4
0.0154
1.644
3862
20
Water
G6P
16
Glycerol
6
Mean B factor (Å2)
PDB Entry
58.67
5UKW
The data in parentheses are for the reflections in the outer resolution shell 2.78–2.65 Å.
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Table 2: Kinetic parameters of HsΔG6PDH mutants.
app
Variable
substrate
kcatapp / Km
Protein
Kmapp (µM) kcatapp (s-1)
IC50DHEA (µM)
11.2 ± 0.6
15.1 ± 0.7
12.3 ± 0.5
(µM-1.s-1)
G6P
54 ± 3
9 ± 1
96 ± 3
17 ± 2
56 ± 3
10.1 ± 0.9
25.0 ± 0.3
19.4 ± 0.7
160 ± 2
0.46 ± 0.02
2.6 ± 0.3
1.67 ± 0.05
9 ± 1
0.37 ± 0.02
1.3 ± 0.1
HsΔG6PDH
NADP+
G6P
HsΔG6PDH_A277C
NADP+
G6P
150 ± 4
21.2 ± 0.3
13.2 ± 0.3
HsΔG6PDH_E347A
NADP+
* Fitted curves are shown at Supporting Material (Fig. S2).
Table 3: Melting Temperature of HsΔG6PDH mutants.
Protein
HsΔG6PDH
HsΔG6PDH_A277C
HsΔG6PDH_E347A
TM (°C)
49.3 ± 0.8
62.2 ± 0.5
49 ± 1
* Raw data and fitted curves are shown at Supporting Material (Fig. S3).
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